The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of intensive study is protein kinases.
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell (see, e.g., G. Hardie et al., The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif., 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids etc). Sequence motifs have been identified that generally correspond to each of these kinase families (see, e.g., S. K. Hanks et al., FASEB J., 1995, 9, 576-596; Knighton et al., Science 1991, 253, 407-414; Hiles et al., Cell, 1992, 70, 419-429; Kunz et al., Cell, 1993, 73, 585-596; Garcia-Bustos et al., EMBO J., 1994, 13, 2352-2361).
In general, protein kinases mediate intracellular signaling by affecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-a), and growth factors (e.g., granulocyte macrophage-colony stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, survival and regulation of the cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, allergies and asthma, Alzheimer's disease and hormone related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.
The Polo-like kinases (PLKs) belong to a family of serine/threonine kinases that are highly conserved across the species, ranging from yeast to man (reviewed in Lowery D M et al., Oncogene, 2005, 24, 248-259). The PLKs have multiple roles in cell cycle, including control of entry into and progression through mitosis.
PLK1 is the best characterized of the PLK family members. PLK1 is widely expressed and is most abundant in tissues with a high mitotic index. Protein levels of PLK1 rise and peak in mitosis (see, e.g., R. Hamanaka et al., J. Biol. Chem., 1995, 270, 21086-21091). The reported substrates of PLK1 are all molecules that are known to regulate entry and progression through mitosis, and include CDC25C, cyclin B, p53, APC, BRCA2 and the proteasome. PLK1 is up-regulated in multiple cancer types and the expression levels correlate with severity of disease (see, e.g., Macmillan, J. C. et al., Ann. Surg. Oncol., 2001, 8, 729-740). PLK1 is an oncogene and can transform NIH-3T3 cells (see, e.g., M. R. Smith et al., Biochem. Biophys. Res. Commun., 1997, 234, 397-405). Depletion or inhibition of PLK1 by siRNA, antisense, microinjection of antibodies, or transfection of a dominant negative construct of PLK1 into cells, reduces proliferation and viability of tumor cells in vitro (see, e.g., R. Guan et al., Cancer Res., 2005, 65, 2698-2704; X. Liu et al., Proc. Nat'l. Acad. Sci. U.S.A., 2003, 100, 5789-5794, Y. Fan et al., World J. Gastroenterol., 2005, 11, 4596-4599; H. A. Lane et al., J. Cell Biol., 1996, 135, 1701-1713). Tumor cells that have been depleted of PLK1 have activated spindle checkpoints and defects in spindle formation, chromosome alignment and separation and cytokinesis. Loss in viability has been reported to be the result of an induction of apoptosis. In contrast, normal cells have been reported to maintain viability on depletion of PLK1. In vivo knock down of PLK1 by siRNA or the use of dominant negative constructs leads to growth inhibition or regression of tumors in xenograft models.
PLK2 is mainly expressed during the G1 phase of the cell cycle and is localized to the centrosome in interphase cells. PLK2 knockout mice develop normally, are fertile and have normal survival rates, but are around 20% smaller than wild type mice. Cells from knockout animals progress through the cell cycle more slowly than in normal mice (see, e.g., S. Ma et al., Mol. Cell. Biol., 2003, 23, 6936-6943). Depletion of PLK2 by siRNA or transfection of kinase inactive mutants into cells blocks centriole duplication. Down-regulation of PLK2 also sensitizes tumor cells to taxol and promotes mitotic catastrophe, in part by suppression of the p53 response (see, e.g., T. F. Burns et al., Mol. Cell. Biol., 2003, 23, 5556-5571).
PLK3 is expressed throughout the cell cycle and increases from G1 to mitosis. Expression is up-regulated in highly proliferating ovarian tumors and breast cancer and is associated with a worse prognosis (see, e.g., W. Weichert et al., Br. J. Cancer, 2004, 90, 815-821; W. Weichert et al., Virchows Arch, 2005, 446, 442-450). In addition to regulation of mitosis, PLK3 is believed to be involved in Golgi fragmentation during the cell cycle and in the DNA-damage response. Inhibition of PLK3 by dominant negative expression is reported to promote p53-independent apoptosis after DNA damage and suppresses colony formation by tumor cells (see, e.g., Z. Li et al., J. Biol. Chem., 2005, 280, 16843-16850).
PLK4 is structurally more diverse from the other PLK family members. Depletion of this kinase causes apoptosis in cancer cells (see, e.g., J. Li et al., Neoplasia, 2005, 7, 312-323). PLK4 knockout mice arrest at E7.5 with a high fraction of cells in mitosis and partly segregated chromosomes (see, e.g., J. W. Hudson et al., Current Biology, 2001, 11, 441-446).
Molecules of the protein kinase family have been implicated in tumor cell growth, proliferation and survival. Accordingly, there is a great need to develop compounds useful as inhibitors of protein kinases. The evidence implicating the PLK kinases as essential for cell division is strong. Blockade of the cell cycle is a clinically validated approach to inhibiting tumor cell proliferation and viability. It would therefore be desirable to develop compounds that are useful as inhibitors of the PLK family of protein kinases (e.g., PLK1, PLK2, PLK3 and PLK4), that would inhibit proliferation and reduce viability of tumor cells, particularly as there is a strong medical need to develop new treatments for cancer, including treatments that would be administered orally.